The invention is directed to a method of determining the presence or absence of cancer or a precancerous condition in epithelial cells, i.e., epithelial cell carcinomas or precancerous epithelium.
Retinoids (vitamin A and its metabolites) can act as chemopreventive and/or chemotherapeutic agents for several types of cancer (Bertram et al., Cancer Res. 1987, 47:3012-3031.; Moon et al., In Sporn, M. B., Roberts, A. B. and Goodman, D. S. (ed.) The Retinoids: Biology, Chemistry and Medicine, 1994, Raven Press, New York, pp. 573-596; Hong et al. In Sporn, M. B., Roberts, A. B. and Goodman, D. S. (ed.) The Retinoids: Biology, Chemistry, and Medicine, 1994, Raven Press, New York, pp. 597-630; Hong et al., Science 1997, 278:1073-1077. Retinoids exert major effects on the growth and differentiation of normal, premalignant, and malignant epithelial cells both in vitro and in vivo (Gudas et al., In Sporn, M. B., Roberts, A. B. and Goodman, D. S. (ed.) The Retinoids: Biology, Chemistry, and Medicine, 1994, Raven Press, New York, pp. 443-520). Retinol can be metabolized to retinyl esters and to various structurally related compounds, such as retinoic acid (RA), retinaldehyde, 4-oxoretinol, 14-hydroxy-4-14-retroretinol (14-HRR), and anhydroretinol in many cell types (Blaner et al., In Sporn, M. B., Roberts, A. B. and Goodman, D. S. (ed.) The Retinoids: Biology, Chemistry, and Medicine, 1994, Raven Press, New York, pp. 229-256; Kurlandsky et al., J. Biol. Chem. 1994, 269:32821-32827; Achkar et al., Proc. Natl. Acad. Sci. USA 1996, 93:4879-4884; Lane et al., Proc. Natl. Acad. Sci. USA 1999, 96:13524-13529; Buck et al., Science 1991, 254:1654-1656; Buck et al, J. Exp. Med. 1993, 178:675-680). While retinoic acid in particular has been demonstrated by many researchers to be useful in the prevention and treatment of cancer in humans (Hong et al., Retinoids in Oncology 1993, Marcel Dekker, New York; Warrell Jr., et al., N. Engl. J. Med 1991, 324:1385-1393), more recently other retinoids such as anhydroretinol have been shown to prevent cancer in animal models (Shealy et al., Oncol. Rep. 1998, 5:857-860).
Retinyl esters are the major metabolites of retinol in some normal cells and tissues, whereas there are data that other cell types are not capable of esterifying retinol. For example, human keratinocytes (Randolph et al. J. Biol. Chem. 1993, 268:9198-9205; Txc3x6rma et al., J. Invest. Dermatol. 1990, 94:132-138; Kurlandsky et al., J. Biol. Chem. 1996, 271:15346-15352; Guo et al., Cancer Res. 1998, 58:166-176; Creek et al. J. Nutr. 1993, 123:356-361; Randolph et al. J. Invest. Dermatol. 1996, 106:168-175), human intestinal Caco-2 cells (Quick et al, Biochemistry 1990, 29:11116-11123.) cultured tracheal epithelial cells (Bhat et al., Biochim. Biophys. Acta 1987, 922:18-27), retinal pigment epithelial cells (Das et al., Biochem J. 1988, 259:459-465; Barry et al., J. Biol. Chem. 1989, 264:9231-9238; Saari et al., J. Biol. Chem. 1989, 264:8636-8640), liver (Ong et al., J. Biol. Chem. 1988, 263:5789-5796; Yost et al., J. Biol. Chem. 1988, 263:18693-18701; Blomhoff et al., J. Biol. Chem. 1985, 260:13560-13565; Matsuura et al., J. Nutr. 1997, 127:218-224; Shimada et al. Arch. Biochem. Biophys. 1997, 344:220-227), and mammary epithelial cells (Ross et al., J. Lipid Res. 1982, 23:133-144; Bhat et al., Cancer Res. 1989, 49:139-144; Chen et al. Cancer Res. 1997, 57:4642-4651) exhibit a high level of retinol esterification activity. Two enzyme activities can catalyze retinyl ester synthesis: acyl CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). The enzyme activities can be distinguished from each other by substrate preferences and differential sensitivities to various inhibitors (Ong et al., J. Biol. Chem. 1988, 263:5789-5796; Yost et al., J. Biol. Chem. 1988, 263:18693-18701; Herr et al. J. Nutr. Biochem. 1991, 503-511). LRAT employs the acyl group at the sn1 position of membrane phospholipid (Herr et al. J. Nutr. Biochem. 1991, 503-511) as an acyl donor, whereas ARAT utilizes acyl CoA (Ross et al., Methods Enzymol. 1990, 189:442-445). ARAT catalyzes esterification of free retinol (Ong et al., J. Biol. Chem. 1988, 263:5789-5796; Yost et al., J. Biol. Chem. 1988, 263:18693-18701; Herr et al. J. Nutr. Biochem. 1991, 503-511; Ong, et al., Nutr. Rev. 1994, 52:S24-S31), while LRAT can utilize both free retinol and retinol bound to the cellular retinol binding protein I as a substrate (Saari et al., Vision Res. 1984, 24:1595-1603). However, it was shown that elevation of the cellular retinol binding protein (CRBP-I) did not enhance retinyl ester storage in transgenic animals (Troen et al., J. Nutr. 1996, 126:2709-2719). An LRAT partial cDNA was recently cloned from human retinal pigment epithelium cells. This cDNA hybridizes to a major RNA transcript of approximately 5.0 kb and minor transcripts of 2.2.-2.5 kb in several tissues, including the testis and liver (Ruiz, et al., J. Biol. Chem. 1999, 274:3834-3841). The ARAT gene has not yet been cloned.
The hydrolysis of retinyl esters also can occur in hepatic cells and in other types of epithelial cells (Cooper, et al, J. Nutr. 1987, 117:2066-2071; Blaner, et al., FEBS Lett. 1990, 274: 89-92; Harrison et al., J. Biol. Chem. 1989, 264:17142-14147; Ghosh, et al. Lipids 1990, 25:221-225; Ritter et al., Biochim. Biophys. Acta 1996, 1291: 228-236; Schlinder, et al, Eur. J. Biochem. 1998, 251:863-873.) Recently, a neutral, bile salt-independent retinyl ester hydrolase (NREH) was purified (Sun, et al. ES-2. J. Biol. Chem. 1997, 272:24488-24493), and a hepatic, bile salt dependent retinyl ester hydrolase was cloned and shown to be identical to pancreatic carboxylester lipase (Chen et al., Proc. Soc. Exp. Biol. Med. 1997, 215:186-191). The retinyl ester hydrolase(s) which are responsible for retinyl ester hydrolysis in many extra-hepatic tissues have not been well characterized, though retinyl ester hydrolases have been described in tissues and cell types in addition to liver. In the retinal pigment epithelium (RPE), all-trans retinyl esters are substrates for an isomerohydrolase which converts the esters into 11-cis retinol; 11-cis retinol is then oxidized and converted to 11-cis retinaldehyde, the chromophore for rhodopsin and cone pigments (Bernstein et al., Proc. Natl. Acad. Sci. USA 1987, 84:1849-1853; Deigner et al., Science 1989, 244:968-971). In adipocytes, there is evidence that retinyl esters can be hydrolyzed by a cyclic AMP dependent enzyme-like hormone sensitive lipase (Wei et al., J. Biol. Chem. 1997, 272:14159-14165).
In contrast to normal epithelial cells, there are some reports that in normal human fibroblasts retinol, although readily taken up by the fibroblasts, is not metabolized to either retinoic acid or retinyl esters (Rundhaug et al., Cancer Res. 1987, 47:5637-5643; Randolph et al., J. Invest. Dermatol. 1998, 111:478-484). In another study, it was reported that cultured human dermal fibroblasts, treated with retinol, metabolized retinol to retinoic acid and retinyl esters (Bailly et al., Exp. Dermatol. 1998, 7:27-34). Little information is available concerning retinol metabolism in normal human endothelial cells. It was previously reported that isolated endothelial cells from the liver contained very low levels of retinoids (Blomhoff et al., J. Biol. Chem. 1985, 260:13560-13565). However, retinoids can influence endothelial cell growth, gene expression, and morphology (Braunhut et al., Microvasc. Res. 1991, 41:47-62; Kooistra et al., J. Biochem. 1995, 232:425-432; Braunhut et al., J. Biol. Chem. 1994, 269:13472-13479; Spencer-Green et al., Clin. Immunol. Immunopath. 1994, 72:53-61; Thompson et al., Eur. J. Biochem. 1991, 203:627-632).
While the functions of retinyl esters are not fully understood, it is believed that retinyl esters act as a storage form for retinol both in the liver and in many other tissues in the body. Interestingly, carcinoma cells of the breast, oral cavity, and skin are deficient in the esterification of retinol (Guo et al., Cancer Res. 1998, 58:166-176; Chen et al. Cancer Res. 1997, 57:4642-4651). These recent data, together with the aforementioned data, suggest that the lack of retinyl esters in carcinoma cells may be associated with or even contribute to their tumorigenic phenotype (Guo et al., supra, Chen et al., supra). However, there is no indication of how this deficiency occurs, or how it could be corrected.
Cancers or malignant tumors are classified according to the type of tissue from which they originate. The broadest division of cancers separates the carcinomas, tumors which arise from epithelial tissues, and the sarcomas, which arise from all other tissues. Epithelium is tissue that covers the internal or external surfaces of the body. Thus, skin, the lining of the mouth, stomach, intestines, bladder and so on are all epithelial tissue.
Within the category of carcinomas, there are many subdivisions, corresponding to the types of different epithelium from which they may be derived. Therefore, the skin, which consists of a type of epithelium called squamous epithelium, can give rise to squamous cell carcinomas. There are other epithelial cells also present in the skin, basal cells, which give rise to basal cell carcinomas, and melanocytes, which give rise to melanomas.
Adenocarcinoma is a cancer originating in glandular cells. Adenocarcinomas occur in the lungs, from small glands in the bronchi; in the stomach from one of the several types of glands lining it; and in the colon, breast, ovaries, testes, prostate and in other locations. Adenocarcinomas arising from different organs can often be identified by the pathologist microscopically, even when they are removed from a different location where they may have metastasized, such as the liver. Thus, it is common to refer to an adenocarcinoma of the stomach which has metastasized to the liver, or one from the colon metastasized to the lungs.
Adenocarcinomas are the most common cell type of cancer, since they include almost all breast cancers, all colon cancers, all prostate cancers, and a fair percentage of lung cancers. The cause of most adenocarcinomas is still unknown, and is the subject of intensive research. Various studies have reported associations of certain carcinomas with other factors such as the association of early menstruation with carcinoma of the breast, or lack of fiber associated with colon cancer. However, these reports change as with fiber and colon cancer (no association) or identify associations that are difficult or impossible change.
Presently, detection of epithelial cell carcinomas involves a pathologist observing cells under a microscope to consider morphology and making a subjective judgment as to whether cancer is present. Thus, there remains a need in the art for objective, accurate methods to determine whether or not an epithelial cell is cancerous or predisposed to become transformed (precancerous condition). The present invention addresses this and other needs in the art.
The instant invention advantageously replaces or supplements the subjective judgment that is now conventional in the diagnosis of hyperplastic, particularly cancerous, conditions with an objective test and minimizes the need for human judgment.
The instant invention is directed to an assay for determining the presence or absence of cancer or the presence of precancerous condition in epithelial cells, and is useful for detecting epithelial cell carcinomas including colon, renal, prostate, oral cavity, lung, breast and skin carcinomas and precancerous conditions associated therewith.
In one embodiment, the invention provides a kit for determining the presence or absence of a cancer or precancerous condition in epithelial cells, which kit comprises an assay for detection of expression levels of LRAT. One preferred LRAT detection assay comprised within the kit is a radiolabeled retinol and an unlabeled retinol, which are used to measure expression levels base on LRAT""s enzymatic activity. Another preferred LRAT detection assay comprised within the kit is a labeled nucleic acid probe or primer that specifically hybridizes to LRAT mRNA, thereby detecting expression of mRNA that is translated into a functional LRAT protein. Yet another preferred LRAT detection assay comprised within the kit is an antibody that specifically binds to LRAT protein in epithelial cells.
In another aspect, the invention provides a method for treating cancer of an epithelial cell or preventing transformation of a precancerous epithelial cell, which method comprises activating LRAT protein expression in the epithelial cell. In a preferred embodiment, LRAT expression is activated by introducing a gene encoding LRAT into the cell.
The invention also provides an expression vector for expression in humans, which vector encodes LRAT under control of an expression control sequence that provides for expression in epithelial cells. In a preferred embodiment, the vector is administered in vivo as an appropriate pharmaceutical composition, where it enters the cells of the organism and mediates expression of the construct.
In its preferred aspect, the invention comprises determining the amount of lecithin:retinol acyltransferase (LRAT) protein present in the cells being tested. Failure to detect LRAT protein indicates presence of cancer, and detection of a lower than normal level of LRAT protein indicates a precancerous condition. It has been discovered that normal epithelial cells contain retinyl esters, whereas cancerous epithelial cells do not, and that this is associated with the presence of LRAT protein in normal epithelial cells but not in cancerous epithelial cells. Abundant levels of LRAT expression are detectable in normal epithelial cells, but LRAT expression is not detected by Western Blot analysis of, or immunohistochemistry studies on, epithelial cells from epithelial cell carcinomas, and intermediate levels of LRAT expression are detected by Western Blot analysis or immunohistochemistry in cells from benign hypertrophy (precancer) epithelial tissues.
These and other aspects of the invention are described in greater detail in the Examples, including Drawings, and Detailed Description, infra.